Original article
European Journal of Microbiology and Immunology 6 (2016) 3, pp. 238–243 DOI: 10.1556/1886.2015.00018
MULTIDRUG RESISTANCE OF ACINETOBACTER BAUMANNII IN LADOKE AKINTOLA UNIVERSITY TEACHING HOSPITAL, OSOGBO, NIGERIA G. Odewale1, O. J. Adefioye1, J. Ojo1, F. A. Adewumi1,2, O. A. Olowe1,* 1
Department of Medical Microbiology and Parasitology, College of Health Sciences, P.M.B. 4400, Ladoke Akintola University of Technology (LAUTECH), Osogbo, Osun State, Nigeria 2 Department of Medical Laboratory Science, Afe Babalola University, P.M.B. 5454, Ado Ekiti, Ekiti State, Nigeria Received: June 2, 2015; Accepted: August 19, 2015 Acinetobacter baumannii is a ubiquitous pathogen that has emerged as a major cause of healthcare-associated infections at Ladoke Akintola University Teaching Hospital. Isolates were assayed according to standard protocol. The isolates were subjected to molecular techniques to detect blaOXA, blaTEM, blaCTX-M, and blaSHV genes in strains of the A. baumannii isolates. The prevalence of A. baumannii was 8.5% and was most prevalent among patients in the age group 51–60 (36%); the male patients (63.6%) were more infected than their female counterparts. Patients (72.7%) in the intensive care unit (ICU) were most infected with this organism. The isolates showed 100% resistance to both amikacin and ciprofloxacin and 90.9% to both ceftriaxone and ceftazidime, while resistance to the other antibiotics used in this study were: piperacillin (81.8%), imipenem (72.7%), gentamycin (72.2%), and meropenem (63.6%). None of the isolates was, however, resistant to colistin. PCR results showed that blaOXA, blaTEM, and blaCTX-M genes were positive in some isolates, while blaSHV was not detected in any of the isolates. This study has revealed that the strains of A. baumannii isolated are multiple drug resistant. Regular monitoring, judicious prescription, and early detection of resistance to these antibiotics are, therefore, necessary to check further dissemination of the organism. Keywords: Acinetobacter baumannii, antibiotic-resistant genes, Nigeria
Introduction Acinetobacter baumannii has been described as one of the most important opportunistic pathogens that cause nosocomial infections in hospitals of the 21st century. This is because of its multiresistant genetic determinants, ability to tolerate a wide range of humidity, pH, salinity, and its survival on many natural sources [1]. The organism’s ability to survive under a wide range of environmental conditions and to persist for extended periods of time on surfaces make it a frequent cause of outbreaks of infections and an endemic health-care-associated pathogen. The aforementioned traits make this pathogen to be ubiquitous in the hospital environment. Outbreaks of A. baumannii colonization and infection in intensive
care units have been described as a common problem [2, 3]. Acinetobacter infections typically occur in very ill patients and can either cause or contribute to death in these patients. They are frequent colonizers of the throat, skin, respiratory tract, and the digestive tract [4] and infect mostly patients with impaired host defenses. The most common clinical presentation of A. baumannii is pneumonia in mechanically ventilated patients in the intensive care units (ICUs) [5] with high mortality rates. Mortality rate for central nervous system (CNS) infection has been revealed to be 20–27% in patients, in whom the organism was isolated from cerebrospinal fluid (CSF) [6]. Other infections caused by this pathogen are bacteraemia, wound infections, secondary meningitis, urinary tract infections, peritonitis, osteomyelitis,
* Corresponding author: O. A. Olowe; Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology, P.M.B. 4400, College of Health Sciences, Isale Osun, Osogbo, Osun State, Nigeria; E-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited. First published online: May 17, 2016
ISSN 2062-8633 © 2016 The Author(s)
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Multidrug resistance of Acinetobacter baumannii
keratitis, and native-valve endocarditis [7]. Invasive procedures involving endotracheal tube, urinary catheter insertions, central venous catheter, ventriculography, myelography, lumbar puncture, exposure to antibiotics, increased length of hospital stay, exposure to patients colonized with A. baumannii, environmental contamination, immunosuppression, emergency admission, respiratory failure or mechanical ventilation, and poor adherence of staff to hand hygiene are the risk factors for the infections caused by A. baumannii [8]. A. baumannii, which was susceptible to antibiotics many years ago [1], is now a multidrug-resistant opportunistic human pathogen that is a frequent cause of nosocomial outbreaks worldwide. A. baumannii strains that are resistant to all major antibiotic classes normally used to treat infections with them, including β-lactams, aminoglycosides, fluoroquinolones, chloramphenicol, tetracycline, and rifampin, are now emerging, and the prevalence of these multidrug-resistant A. baumannii strains leaves limited clinical options for treatment [1], underscoring the need to develop novel antibiotics for bacterial pathogens in general and gram-negative pathogens in particular. Although nosocomial infections caused by A. baumannii have been reported worldwide [9], very little has been reported on the prevalence and antibiotic susceptibility of A. baumannii isolates in Nigeria. The aim of this work was, therefore, to assess the prevalence and the current level of antimicrobial resistance pattern of the isolates in Ladoke Akintola University of Technology (LAUTECH) Teaching Hospital.
Identification of Acinetobacter baumannii from clinical specimens Samples from the patients were cultivated onto MacConkey agar and incubated at 37 °C for 24–48 h under aerobic conditions. Acinetobacter species were primarily identified on the basis of their Gram’s reaction and cultural characteristics, further biochemical tests were done with the aid of the API 20NE (Bio Merieux, France) to identify their different strains. Antimicrobial susceptibility testing Antimicrobial susceptibility of the isolates to imipenem, meropenem, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, colistin, and ceftriazone which were obtained from Oxoid Company, UK was determined by using the disc diffusion method. The results were interpreted according to the manufacturer’s instructions and CLSI guidelines [10]. Amplification of extended spectrum β-lactamase genes Polymerase chain reaction (PCR) was used to detect genes encoding resistance to the extended spectrum β-lactams: blaOXA, blaTEM, blaCTX-M, and blaSHV according to the method described by Olowe et al. [11]. The list of primers that were used is shown in Table 1. Data analysis
Materials and methods Data are presented as frequencies and percentages. Statistical analysis was performed using the chi-square (χ2) test.
Collection of clinical specimens Tracheal aspirate, blood, urine, sputum, CSF, and wound swabs of 150 patients (89 male and 61 female) were collected aseptically and transported immediately to the research laboratory for microbiological analysis. The study population comprised of patients who have had surgery, on ventilators or intubation, and prior history of antibiotic use.
Ethical approval Clearance of research involving human subjects was granted by the Ethics and Research Committee of the Ladoke Akintola University Teaching Hospital, Osogbo, Nigeria for this study.
Table 1. Primers used for the amplification of genes Primer
Sequence 51–31
OXA-F
GCGTGGTTAAGGATGAACAC
OXA-R
CATCAAGTTCAACCCAACCG
CTX-M F
CGATGTGCAGTACCAGTAA
CTX-M R
TTAGTGACCAGAATAAGCGG
TEM F
CCCCGAAGAACGTTTTC
TEM R
ATCAGCAATAAACCAGC
SHV F
AGGATTGACTGCCTTTTTG
SHV R
ATTTGCTGATTTCGCTCG
Gene
Product size (bp)
Annealing temp. (°C)
blaOXA
620
56
blaCTX-M
585
60
blaTEM
517
51
blaSHV
393
56
European Journal of Microbiology and Immunology
240
G. Odewale et al.
Results Majority of the A. baumannii-positive patients, 10 (90.9%), were in the age bracket 41–70 (Table 2). The blood sample of a 1-year-old patient who had malaria sepsis was also positive for this organism. The male patients, 7 (63.6%), were more positive for A. baumannii than their female counterparts, 4 (36.4%) (Table 2). A larger percentage of the isolates, 8 (72.7%), were isolated from the ICU, 2 (18.2%) from the surgical wards, and 1 (9.1%) from the paediatric wards (Table 2). The risk factors for the acquisition of A. baumannii infection were age >40 years and
European Journal of Microbiology and Immunology 6 (2016) 3, pp. 238–243 DOI: 10.1556/1886.2015.00018
MULTIDRUG RESISTANCE OF ACINETOBACTER BAUMANNII IN LADOKE AKINTOLA UNIVERSITY TEACHING HOSPITAL, OSOGBO, NIGERIA G. Odewale1, O. J. Adefioye1, J. Ojo1, F. A. Adewumi1,2, O. A. Olowe1,* 1
Department of Medical Microbiology and Parasitology, College of Health Sciences, P.M.B. 4400, Ladoke Akintola University of Technology (LAUTECH), Osogbo, Osun State, Nigeria 2 Department of Medical Laboratory Science, Afe Babalola University, P.M.B. 5454, Ado Ekiti, Ekiti State, Nigeria Received: June 2, 2015; Accepted: August 19, 2015 Acinetobacter baumannii is a ubiquitous pathogen that has emerged as a major cause of healthcare-associated infections at Ladoke Akintola University Teaching Hospital. Isolates were assayed according to standard protocol. The isolates were subjected to molecular techniques to detect blaOXA, blaTEM, blaCTX-M, and blaSHV genes in strains of the A. baumannii isolates. The prevalence of A. baumannii was 8.5% and was most prevalent among patients in the age group 51–60 (36%); the male patients (63.6%) were more infected than their female counterparts. Patients (72.7%) in the intensive care unit (ICU) were most infected with this organism. The isolates showed 100% resistance to both amikacin and ciprofloxacin and 90.9% to both ceftriaxone and ceftazidime, while resistance to the other antibiotics used in this study were: piperacillin (81.8%), imipenem (72.7%), gentamycin (72.2%), and meropenem (63.6%). None of the isolates was, however, resistant to colistin. PCR results showed that blaOXA, blaTEM, and blaCTX-M genes were positive in some isolates, while blaSHV was not detected in any of the isolates. This study has revealed that the strains of A. baumannii isolated are multiple drug resistant. Regular monitoring, judicious prescription, and early detection of resistance to these antibiotics are, therefore, necessary to check further dissemination of the organism. Keywords: Acinetobacter baumannii, antibiotic-resistant genes, Nigeria
Introduction Acinetobacter baumannii has been described as one of the most important opportunistic pathogens that cause nosocomial infections in hospitals of the 21st century. This is because of its multiresistant genetic determinants, ability to tolerate a wide range of humidity, pH, salinity, and its survival on many natural sources [1]. The organism’s ability to survive under a wide range of environmental conditions and to persist for extended periods of time on surfaces make it a frequent cause of outbreaks of infections and an endemic health-care-associated pathogen. The aforementioned traits make this pathogen to be ubiquitous in the hospital environment. Outbreaks of A. baumannii colonization and infection in intensive
care units have been described as a common problem [2, 3]. Acinetobacter infections typically occur in very ill patients and can either cause or contribute to death in these patients. They are frequent colonizers of the throat, skin, respiratory tract, and the digestive tract [4] and infect mostly patients with impaired host defenses. The most common clinical presentation of A. baumannii is pneumonia in mechanically ventilated patients in the intensive care units (ICUs) [5] with high mortality rates. Mortality rate for central nervous system (CNS) infection has been revealed to be 20–27% in patients, in whom the organism was isolated from cerebrospinal fluid (CSF) [6]. Other infections caused by this pathogen are bacteraemia, wound infections, secondary meningitis, urinary tract infections, peritonitis, osteomyelitis,
* Corresponding author: O. A. Olowe; Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology, P.M.B. 4400, College of Health Sciences, Isale Osun, Osogbo, Osun State, Nigeria; E-mail: [email protected] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited. First published online: May 17, 2016
ISSN 2062-8633 © 2016 The Author(s)
239
Multidrug resistance of Acinetobacter baumannii
keratitis, and native-valve endocarditis [7]. Invasive procedures involving endotracheal tube, urinary catheter insertions, central venous catheter, ventriculography, myelography, lumbar puncture, exposure to antibiotics, increased length of hospital stay, exposure to patients colonized with A. baumannii, environmental contamination, immunosuppression, emergency admission, respiratory failure or mechanical ventilation, and poor adherence of staff to hand hygiene are the risk factors for the infections caused by A. baumannii [8]. A. baumannii, which was susceptible to antibiotics many years ago [1], is now a multidrug-resistant opportunistic human pathogen that is a frequent cause of nosocomial outbreaks worldwide. A. baumannii strains that are resistant to all major antibiotic classes normally used to treat infections with them, including β-lactams, aminoglycosides, fluoroquinolones, chloramphenicol, tetracycline, and rifampin, are now emerging, and the prevalence of these multidrug-resistant A. baumannii strains leaves limited clinical options for treatment [1], underscoring the need to develop novel antibiotics for bacterial pathogens in general and gram-negative pathogens in particular. Although nosocomial infections caused by A. baumannii have been reported worldwide [9], very little has been reported on the prevalence and antibiotic susceptibility of A. baumannii isolates in Nigeria. The aim of this work was, therefore, to assess the prevalence and the current level of antimicrobial resistance pattern of the isolates in Ladoke Akintola University of Technology (LAUTECH) Teaching Hospital.
Identification of Acinetobacter baumannii from clinical specimens Samples from the patients were cultivated onto MacConkey agar and incubated at 37 °C for 24–48 h under aerobic conditions. Acinetobacter species were primarily identified on the basis of their Gram’s reaction and cultural characteristics, further biochemical tests were done with the aid of the API 20NE (Bio Merieux, France) to identify their different strains. Antimicrobial susceptibility testing Antimicrobial susceptibility of the isolates to imipenem, meropenem, ceftazidime, piperacillin, amikacin, gentamicin, ciprofloxacin, colistin, and ceftriazone which were obtained from Oxoid Company, UK was determined by using the disc diffusion method. The results were interpreted according to the manufacturer’s instructions and CLSI guidelines [10]. Amplification of extended spectrum β-lactamase genes Polymerase chain reaction (PCR) was used to detect genes encoding resistance to the extended spectrum β-lactams: blaOXA, blaTEM, blaCTX-M, and blaSHV according to the method described by Olowe et al. [11]. The list of primers that were used is shown in Table 1. Data analysis
Materials and methods Data are presented as frequencies and percentages. Statistical analysis was performed using the chi-square (χ2) test.
Collection of clinical specimens Tracheal aspirate, blood, urine, sputum, CSF, and wound swabs of 150 patients (89 male and 61 female) were collected aseptically and transported immediately to the research laboratory for microbiological analysis. The study population comprised of patients who have had surgery, on ventilators or intubation, and prior history of antibiotic use.
Ethical approval Clearance of research involving human subjects was granted by the Ethics and Research Committee of the Ladoke Akintola University Teaching Hospital, Osogbo, Nigeria for this study.
Table 1. Primers used for the amplification of genes Primer
Sequence 51–31
OXA-F
GCGTGGTTAAGGATGAACAC
OXA-R
CATCAAGTTCAACCCAACCG
CTX-M F
CGATGTGCAGTACCAGTAA
CTX-M R
TTAGTGACCAGAATAAGCGG
TEM F
CCCCGAAGAACGTTTTC
TEM R
ATCAGCAATAAACCAGC
SHV F
AGGATTGACTGCCTTTTTG
SHV R
ATTTGCTGATTTCGCTCG
Gene
Product size (bp)
Annealing temp. (°C)
blaOXA
620
56
blaCTX-M
585
60
blaTEM
517
51
blaSHV
393
56
European Journal of Microbiology and Immunology
240
G. Odewale et al.
Results Majority of the A. baumannii-positive patients, 10 (90.9%), were in the age bracket 41–70 (Table 2). The blood sample of a 1-year-old patient who had malaria sepsis was also positive for this organism. The male patients, 7 (63.6%), were more positive for A. baumannii than their female counterparts, 4 (36.4%) (Table 2). A larger percentage of the isolates, 8 (72.7%), were isolated from the ICU, 2 (18.2%) from the surgical wards, and 1 (9.1%) from the paediatric wards (Table 2). The risk factors for the acquisition of A. baumannii infection were age >40 years and
